连续三年夜间增温和施氮对云杉外生菌根及菌根真菌多样性的影响

以西南亚高山针叶林建群种粗枝云杉(Picea asperata)为研究对象,采用红外加热模拟增温结合外施氮肥(NH₄NO₃ 25 g N m^-2 a^-1)的方法,研究连续3a夜间增温和施肥对云杉幼苗外生菌根侵染率、土壤外生菌根真菌生物量及其群落多样性的影响。结果表明:夜间增温对云杉外生菌根侵染率的影响具有季节性及根级差异。夜间增温对春季(2011年5月)云杉1级根,夏季(2011年7月)和秋季(2010年10月)云杉2级根侵染率影响显著。除2011年7月1级根外,施氮对云杉1、2级根侵染率无显著影响。夜间增温对土壤中外生菌根真菌的生物量和群落多样性无显著影响,施氮及增温与施氮联合处理使土壤中外生菌根真菌生物量显著降低,但却提高了外生菌根真菌群落的多样性。这说明云杉幼苗外生菌根侵染率对温度较敏感,土壤外生菌根真菌生物量及其群落多样性对施氮较敏感。这为进一步研究该区域亚高山针叶林地下过程对全球气候变化的响应机制提供了科学依据。     

Chitin and ergosterol combined to measure total and living fungal biomass in ectomycorrhizas

We have studied the chitin and ergosterol contents of ectomycorrhizal roots in three sets of experiments to evaluate them as indicators of fungal biomass. The first set of experiments showed that ageing had a marked effect on ergosterol concentrations. The ergosterol content of 7-month-old, brown, shrunken Pinus sylvestris L.– Paxillus involutus (Fr.) Fr. mycorrhizas was found to be only 10% of that found in white, turgid, 1- or 4-month-old specimens. This supports the hypothesis that the compound is a good indicator of living fungal biomass. Ageing had a lesser effect on chitin concentrations since the chitin levels found in 7-month-old mycorrhizas were still 60% of the levels found in 1- and 4-month-old specimens.
Consequently, the chitin:ergosterol ratio increased from about 14 to 19 in 1- and 4-month-old mycorrhizas respectively to about 110 in 7-month-old mycorrhizas. In the second set of experiments, we found that variation in plant growth had no effect on the chitin:ergosterol ratio in whole root systems of either Alnus incana (L.) Moench or Pinus sylvestris mycorrhizal with Paxillus involutus . In the third set of experiments, we found a constant relationship between the two marker concentrations in 10-month-old root systems of Pinus sylvestris , regardless of fungal species involved, using Paxillus involutus , Piloderma croceum Erikss. & Hjorts and Suillus variegatus (Fr.) O. Kuntze as test organisms. Taken together, the results of this study suggest that both chitin and ergosterol give reliable, but different, relative measures of fungal biomass in mycorrhizal roots. Furthermore, we demonstrate that, in combination, the two chemical markers can be used to estimate both total and living fungal biomass (derived from the chitin:ergosterol ratio).     

Nitrogen isotopes in ectomycorrhizal sporocarps correspond to belowground exploration types

Nitrogen isotope values (δ¹⁵N) are higher in ectomycorrhizal fungi than in their plant hosts but the wide variability in δ¹⁵N among sporocarps of different fungal taxa is unexplained. We propose that fungal δ¹⁵N reflects sequestration of fungal nitrogen to build fungal biomass, and should accordingly reflect fungal exploration strategies and hyphal properties. To test this, we compared δ¹⁵N to exploration types, hyphal hydrophobicity, and the presence of rhizomorphs in ectomycorrhizal species from surveys at four sites in temperate and boreal coniferous forests. Fungi with exploration types of high biomass, such as long-distance (e.g., Suillus), medium-distance mat (e.g., Hydnellum), and medium-distance fringe (e.g., Cortinarius) were 4–7‰ more enriched in ¹⁵N than fungi with exploration types of low biomass [medium-distance smooth (e.g., Amanita), short-distance (e.g., Inocybe), and contact (e.g., Hygrophorus)]. High biomass types comprised 79% (Åheden, northern Sweden), 65% (Deer Park, Pacific Northwest, USA), 45% (Stadsskogen, central Sweden), and 39% (Hoh, Pacific Northwest, USA) of ectomycorrhizal species, with these types more prevalent at sites of lower nitrogen availability. Species with hydrophobic hyphae or with rhizomorphs were 3–4‰ more enriched in ¹⁵N than taxa with hydrophilic hyphae or without rhizomorphs. The consistency of these patterns suggest that δ¹⁵N measurements could provide insights into belowground functioning of poorly known taxa of ectomycorrhizal fungi and into relative fungal biomass across ectomycorrhizal communities.     

Ectomycorrhizal fungal diversity and saprotrophic fungal diversity are linked to different tree community attributes in a field‐based tree experiment

Exploring the link between above‐ and belowground biodiversity has been a major theme of recent ecological research, due in large part to the increasingly well‐recognized role that soil microorganisms play in driving plant community processes. In this study, we utilized a field‐based tree experiment in Minnesota, USA, to assess the effect of changes in plant species richness and phylogenetic diversity on the richness and composition of both ectomycorrhizal and saprotrophic fungal communities. We found that ectomycorrhizal fungal species richness was significantly positively influenced by increasing plant phylogenetic diversity, while saprotrophic fungal species richness was significantly affected by plant leaf nitrogen content, specific root length and standing biomass. The increasing ectomycorrhizal fungal richness associated with increasing plant phylogenetic diversity was driven by the combined presence of ectomycorrhizal fungal specialists in plots with both gymnosperm and angiosperm hosts. Although the species composition of both the ectomycorrhizal and saprotrophic fungal communities changed significantly in response to changes in plant species composition, the effect was much greater for ectomycorrhizal fungi. In addition, ectomycorrhizal but not saprotrophic fungal species composition was significantly influenced by both plant phylum (angiosperm, gymnosperm, both) and origin (Europe, America, both). The phylum effect was caused by differences in ectomycorrhizal fungal community composition, while the origin effect was attributable to differences in community heterogeneity. Taken together, this study emphasizes that plant‐associated effects on soil fungal communities are largely guild‐specific and provides a mechanistic basis for the positive link between plant phylogenetic diversity and ectomycorrhizal fungal richness.     

Dramatic changes in ectomycorrhizal community composition, root tip abundance and mycelial production along a stand-scale nitrogen deposition gradient

? Nitrogen (N) availability is known to influence ectomycorrhizal fungal components, such as fungal community composition, biomass of root tips and production of mycelia, but effects have never been demonstrated within the same forest. ? We measured concurrently the abundance of ectomycorrhizal root tips and the production of external mycelia, and explored the changes in the ectomycorrhizal community composition, across a stand-scale N deposition gradient (from 27 to 43 kg N ha?1 yr?1) at the edge of a spruce forest. The N status was affected along the gradient as shown by a range of N availability indices. ? Ectomycorrhizal root tip abundance and mycelial production decreased five and 10-fold, respectively, with increasing N deposition. In addition, the ectomycorrhizal fungal community changed and the species richness decreased. The changes were correlated with the measured indices of N status, in particular N deposition and N leaching. ? The relationship between the altered ectomycorrhizal community, root tip abundance and mycelial production is discussed in the context of the N parameters. We suggest that increased N deposition to forests will cause large changes in ectomycorrhizal fungal community structure and functioning, which, in turn, may result in reduced N uptake by roots and fungi, and increased losses of N by leaching.     

Identification of genes differentially expressed in extraradical mycelium and ectomycorrhizal roots during Paxillus involutus-Betula pendula ectomycorrhizal symbiosis

The development of ectomycorrhizal symbiosis leads to drastic changes in gene expression in both partners. However, little is known about the spatial regulation of symbiosis-regulated genes. Using cDNA array profiling, we compared the levels of expression of fungal genes corresponding to approximately 1,200 expressed sequenced tags in the ectomycorrhizal root tips (ECM) and the connected extraradical mycelium (EM) for the Paxillus involutus-Betula pendula ectomycorrhizal association grown on peat in a microcosm system. Sixty-five unique genes were found to be differentially expressed in these two fungal compartments. In ECM, a gene coding for a putative phosphatidylserine decarboxylase (Psd) was up-regulated by 24-fold, while genes coding for urea (Dur3) and spermine (Tpo3) transporters were up-regulated 4.1- and 6.2-fold in EM. Moreover, urea was the major nitrogen compound found in EM by gas chromatography-mass spectrometry analysis. These results suggest that (i) there is a spatial difference in the patterns of fungal gene expression between ECM and EM, (ii) urea and polyamine transporters could facilitate the translocation of nitrogen compounds within the EM network, and (iii) fungal Psd may contribute to membrane remodeling during ectomycorrhiza formation.     

The potential for mycobiont sharing between shrubs and seedlings to facilitate tree establishment after wildfire at Alaska arctic treeline

Root‐associated fungi, particularly ectomycorrhizal fungi (EMF), are critical symbionts of all boreal tree species. Although climatically driven increases in wildfire frequency and extent have been hypothesized to increase vegetation transitions from tundra to boreal forest, fire reduces mycorrhizal inoculum. Therefore, changes in mycobiont inoculum may potentially limit tree‐seedling establishment beyond current treeline. We investigated whether ectomycorrhizal shrubs that resprout after fire support similar fungal taxa to those that associate with tree seedlings that establish naturally after fire. We then assessed whether mycobiont identity correlates with the biomass or nutrient status of these tree seedlings. The majority of fungal taxa observed on shrub and seedling root systems were EMF, with some dark septate endophytes and ericoid mycorrhizal taxa. Seedlings and adjacent shrubs associated with similar arrays of fungal taxa, and there were strong correlations between the structure of seedling and shrub fungal communities. These results show that resprouting postfire shrubs support fungal taxa compatible with tree seedlings that establish after wildfire. Shrub taxon, distance to the nearest shrub and fire severity influenced the similarity between seedling and shrub fungal communities. Fungal composition was correlated with both foliar C:N ratio and seedling biomass and was one of the strongest explanatory variables predicting seedling biomass. While correlative, these results suggest that mycobionts are important to nutrient acquisition and biomass accrual of naturally establishing tree seedlings at treeline and that mycobiont taxa shared by resprouting postfire vegetation may be a significant source of inoculum for tree‐seedling establishment beyond current treeline.     

Elevated CO2 impacts ectomycorrhiza-mediated forest soil carbon flow: fungal biomass production,respiration and exudation

Large quantities of carbon are exchanged between terrestrial ecosystems and the atmosphere, and extensive research efforts are made to understand carbon cycling and the impact of elevated atmospheric CO₂ levels. The response of soils to increased carbon availability is largely driven by root associated ectomycorrhizal fungi in forest ecosystems, since they partition host derived carbon belowground. In this review I examine how CO₂ enrichment affects ectomycorrhizal fungal biomass production, exudation, respiration, soil carbon fluxes, and other soil microbes, and the importance of the fungal species in these responses. I briefly discuss the significance of CO₂ alterations in the mycorrhizal symbiosis in the context of consequences for carbon sequestration, and present research priorities.     

The extraction and quantification of ergosterol from ectomycorrhizal fungi and roots

Recently, ergosterol analysis has been used to quantify viable fungal biomass in resynthesized ectomycorrhizae. An objective of our study was to quantify ergosterol in a range of ectomycorrhizal isolates under differing growth conditions. In addition, we tested the applicability of the method on field-collected roots of ectomycorrhizal and vesicular-arbuscular (VA) mycorrhizal plants. Quantification of sitosterol as a biomass indicator of plant roots was also undertaken. Ergosterol was not detected in roots of uninoculated Betula populifolia seedlings, and sitosterol was not detected in an ectomycorrhizal fungal isolate but was present in birch roots. Ergosterol was produced in all isolates examined, which represented the major orders of ectomycorrhizal fungi. The range of values obtained, from 3 to nearly 18 g ergosterol mg^-1 dry mass, agrees well with reported values for other mycorrhizal and decomposer fungi. Hyphal ergosterol was the same during growth on phytic acid and KH₂PO₄. Reduction of growth temperature from 25° C to 15° C had little effect on ergosterol content of cultures harvested at similar growth stages. Ergosterol and sitosterol were detected in field-collected ectomycorrhizae of B. populifolia and Pinus sylvestris and VA mycorrhizae of Acer rubrum and Plantago major. Both ergosterol content and ergosterol to sitosterol ratios were significantly lower in VA mycorrhizae than ectomycorrhizae. Calculations of viable fungal biomass associated with field-collected roots were in agreement with those reported by others using the method on resynthesized ectomycorrhizae. Estimates of total mass could be obtained for field-collected B. populifolia roots by a simultaneously using ergosterol to estimate fungal biomass and sitosterol to estimate root mass. Some potential applications and limitations of sterol quantification in studies of mycorrhizal physiology and ecology are discussed.     

Identification of Genes Differentially Expressed in Extraradical Mycelium and Ectomycorrhizal Roots during Paxillus involutus-Betula pendula Ectomycorrhizal Symbiosis

The development of ectomycorrhizal symbiosis leads to drastic changes in gene expression in both partners. However, little is known about the spatial regulation of symbiosis-regulated genes. Using cDNA array profiling, we compared the levels of expression of fungal genes corresponding to approximately 1,200 expressed sequenced tags in the ectomycorrhizal root tips (ECM) and the connected extraradical mycelium (EM) for the Paxillus involutus-Betula pendula ectomycorrhizal association grown on peat in a microcosm system. Sixty-five unique genes were found to be differentially expressed in these two fungal compartments. In ECM, a gene coding for a putative phosphatidylserine decarboxylase (Psd) was up-regulated by 24-fold, while genes coding for urea (Dur3) and spermine (Tpo3) transporters were up-regulated 4.1- and 6.2-fold in EM. Moreover, urea was the major nitrogen compound found in EM by gas chromatography-mass spectrometry analysis. These results suggest that (i) there is a spatial difference in the patterns of fungal gene expression between ECM and EM, (ii) urea and polyamine transporters could facilitate the translocation of nitrogen compounds within the EM network, and (iii) fungal Psd may contribute to membrane remodeling during ectomycorrhiza formation.     

Determination of chitin in fungi and mycorrhizal roots by an improved HPLC analysis of glucosamine

A method to measure chitin content in fungi and ectomycorrhizal roots with high-performance liquid chromatography (HPLC) was developed. Measurements of fluorescence of 9-fluorenylmethylchloroformate (FMOC-CI) derivatives of glucosamine were made on acid hydrolysates of pure chitin, chitin-root mixtures and fungal-root mixtures. The method was applied on 5 isolates of ectomycorrhizal fungi, and ectomycorrhizal and non-mycorrhizal Pinus sylvestris roots. Interference from amino acids was removed by pre-treatment of samples with 0.2 N NaOH. This pre-treatment did not reduce the recovery of chitin, nor did plant material affect the recovery of chitin. The HPLC method was compared with a colorimetric chitin-method by measurements on root-fungal mixtures, with known fungal content. The HPLC method gave estimates of fungal biomass which were equal to the expected while the colorimetric method showed values significantly (p<0.001) lower than the expected. The present chitin method offers a sensitive and specific tool for the quantification of chitin in fungi and in ectomycorrhizal roots.     

The annual invasive plant Impatiens glandulifera reduces hyphal biomass of soil fungi in deciduous forests

Soil fungi play a crucial role in ecosystem functioning and there is increasing evidence that exotic plants invading forests can affect soil fungal communities. We examined potential effects of the invasive plant Impatiens glandulifera on hyphal biomass of ectomycorrhizal fungi, their genetic diversity and the diversity of other soil fungi in deciduous forests in Switzerland. We compared invaded patches with patches where I. glandulifera had been removed, by establishing pairs of 3-m long transect lines at the edge of seven areas of either type. Along the transects we assessed the length of ectomycorrhizal fungal hyphae using the ‘ingrowth mesh bag method’, and used terminal restriction fragment length polymorphism (T-RFLP) analysis to examine fungal genetic diversity. The invasive plant reduced fungal hyphal biomass by 30–80%: the reduction was largest in the centre of the patch. I. glandulifera did not alter fungal richness, but affected the composition of fungal communities. This is probably the result of a decrease of mycorrhizal fungi, coupled with an increase of saprotrophic fungi. Our findings demonstrate the adverse impacts of an annual invasive plant species on both fungal hyphal biomass and the composition of soil fungal communities. This may negatively affect forest nutrient and carbon cycling, soil stability and the functionality of the fungal community, with major consequences for forest ecosystem functioning.     

Fungal content of ectomycorrhizal tips: comparison among 13 tree species

To better understand soil carbon cycling in forest ecosystems, we studied the proportion of fungal sheath area (FSA) in the cross-sectional ectomycorrhizal area in 13 tree species. Ectomycorrhizal samples were collected from subalpine and temperate forests in Japan. The FSA values were in the range of 12% to 56% across all tree species, tree ages, and fungal species. In Abies firma and Quercus serrata, the FSA values were larger in mature trees than in seedlings, whereas no such differences were found in Pinus densiflora and Fagus crenata. In broad-leaved trees, because the plant tissue radii lay within a narrow range, the FSA was affected mainly by the fungal sheath thickness. In conifers, however, the plant tissue radii varied widely among genera, so the FSA was affected by both the plant tissue radius and the fungal sheath thickness. Our findings suggest that the fungal content of ectomycorrhizal tips differs among tree species and fungal species, so that both parameters must be considered in studies of forest carbon cycling. The estimates revealed that data gathering in each type of forest leads to more accurate estimates of the biomass of fungi in ectomycorrhizal tips.     

Ectomycorrhizal fungal biomass in roots and uptake of P from apatite by Pinus sylvestris seedlings growing in forest soil with and without wood ash amendment

Forest soil from an experimental Norway spruce forest with four levels of wood ash addition (0, 1, 3 and 6 tonnes ha^–1) was used to inoculate pine (Pinus sylvestris) seedlings with indigenous ectomycorrhizal (EM) fungi. Uptake of ³²P and ⁸⁶Rb in a root bioassay was used to estimate the demand for P and K by seedlings grown in the different soils. Utilisation of P from apatite was tested in a laboratory system where uptake by the ectomycorrhizal mycelium was separated from uptake by roots. The demand for P and K in the seedlings was similar regardless of the ash treatment. Variation in EM levels, estimated as fungal biomass (ergosterol) in roots, was large in the different soils, but not related to ash addition. Uptake of P from apatite was, on average, 23% of total seedling P and was not related to EM levels. It was concluded that the improved P uptake from apatite by EM fungi found in earlier studies is probably not a general phenomenon among EM fungi. The small effect of ash addition on EM levels and P uptake suggests that addition of granulated wood ash is a forest management treatment that will have only minor influence on ectomycorrhizal symbiosis.     

Multiple-host fungi are the most frequent and abundant ectomycorrhizal types in a mixed stand of Douglas fir (Pseudotsuga menziesii) and bishop pine (Pinus muricata)

The ectomycorrhizal fungal associations of Douglas fir ( Pseudotsuga menziesii D. Don) and bishop pine ( Pinus muricata D. Don) were investigated in a mixed forest stand. We identified fungi directly from field-collected ectomycorrhizal (ECM) root tips using PCR-based methods. Sixteen species of fungi were found, of which twelve associated with both hosts. Rhizopogon parksii Smith was specific to Douglas fir. Three other species colonized only one of the hosts, but were too infrequent to draw conclusions about specificity. Seventy-four percent of the biomass of ECM root tips sampled in the stand were colonized by members of the Thelephoraceae and Russulaceae. All 12 species of fungi that associated with both tree species did so within a 10×40 cm soil volume, suggesting that individual fungal genotypes linked the putatively competing tree hosts.